The invention relates to the field of therapeutics and diagnostics, and in particular to the delivery of biological molecules and other chemical substances into the interior of cells.
Many biological molecules and pharmaceutical agents must first traverse the cell membrane in order to exert an action on cells. Transmembrane delivery of nucleic acids, for example, has relied on protein carriers, antibody carriers, liposomal delivery systems, direct injection into cells, electroporation, cell fusion, viral delivery, and calcium-phosphate mediated transformation.
Another method of transmembrane delivery of exogenous molecules, including nucleic acids, has been receptor-mediated endocytosis. This involves conjugating the biological or pharmaceutical agent with a ligand which specifically binds to receptors on a cell membrane. The process of endocytosis is initiated or activated by the binding of the ligand to the receptor. Receptor-mediated endocytosis has been utilized for delivery of proteins as well as nucleic acids to cells. Generally, the ligand is chemically conjugated by covalent, ionic or hydrogen bonding to the exogenous molecule of interest that is still recognized in the conjugate by the target receptor. Conjugation of exogenous molecules of interest to ligand substances having corresponding cell surface receptors is described in U.S. Pat. No. 5,108,921. In particular, the method of the ""921 patent relies upon the transmembrane transport of exogenous materials across a membrane having biotin or folate receptors that initiate transmembrane transport of receptor bound species.
International patent application PCT/US90/01002 (WO 90/104448) of Genentech, Inc., discloses covalent conjugates of oligonucleotides and lipids for securing transmembrane delivery of the oligonucleotide into cells. Examples of such lipids include fatty acids and esters thereof, glycerides, e.g., triglycerides, glyceryl ethers, phospholipids, sphingolipids, fatty alcohols, waxes, terpenes, and steroids. The lipids may be naturally derived or synthetically prepared.
International patent application PCT/US90/05272 (WO 91/04753) of Cetus Corporation, describes conjugates of antisense oligonucleotides and ligand-binding molecules which recognize a cell surface receptor. The ligand-binding molecule is a growth factor, an antibody to a growth factor, or an antibody to a cell surface receptor.
U.S. Pat. No. 5,550,111 discloses conjugates of 2xe2x80x2, 5xe2x80x2-oligoadenylate and an adduct which results in enhanced penetration into cells. The adduct may comprise a vitamin selected from those vitamins which have a corresponding cell receptor on targeted mammalian cells. Such vitamins include for example, vitamin B12, biotin, riboflavin or folic acid. Alternatively, the adduct may comprise a lipophilic molecule or radical, such as an acyl group of the formula xe2x80x94OC(CH2)xCH3, wherein x is an integer from 1 to 20, preferably from 2 to 14.
Frequently, useful effectors of intracellular targets comprise proteinaceous substances such as peptides and polypeptides. For example, the product of the Bcl-2 gene is known to contribute to neoplastic cell expansion by preventing normal cell turnover caused by physiological cell death mechanisms. The Bcl-2 gene product is an intracellular protein. Bcl-2 (B cell lymphomalleukemia 2) was originally identified at the chromosomal breakpoint of t(14;18)-bearing B-cell lymphomas. Bcl-2 is now known to belong to a growing family of proteins which regulate programmed cell death or apoptosis. The Bcl-2 family includes both death antagonists (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, Brag-l, Mcl-l and Al) and death agonists (Bax, Bak, Bcl-x5, Bad, Bid, Bik and Hrk) (Thompson, Science 267:1456-62 (1992); Reed, J. Cell Biol. 124:1-6 (1994); Yang et al., Blood 88:386401 (1996)). This family of molecules shares four homologous regions termed Bcl homology (BH) domains BH1, BH2, BH3, and BH4. All death antagonist members contain the BH4 domain while the agonist members lack BH4. It is known that the BH1 and BH2 domains of the death antagonists such as Bcl-2 are required for these proteins to heterodimerize with death agonists, such as Bax, and to repress cell death. On the other hand, the BH3 domain of death agonists is required for these proteins to heterodimerize with Bcl-2 and to promote apoptosis.
Programmed cell death or apoptosis plays a fundamental role in the development and maintenance of cellular homeostasis. Homologous proteins and pathways in apoptosis are found in a wide range of species, indicating that cellular demise is critical for the life and death cycle of the cell in all organisms. When extracellular stimuli switch on the cell-death signal, the response of the cell to such stimuli is specific for the particular cell type (Bonini et al., Cell 72:379-95 (1993)). The pathway to cellular suicide is controlled by certain checkpoints (Oltvai, Cell 79:189-92 (1994)). The Bcl family proteins, including both antagonists of apoptosis (such as Bcl-2) and agonists of apoptosis (such as Bax), constitute the primary checkpoint. As such, the transmission of a cell-death signal can be either promoted or blocked by the different combinations of the Bcl-2 family members. The three-dimensional structure of a death antagonist, Bcl-XL, as determined by X-ray crystallography and NMR spectroscopy, provides a structural basis for understanding the biological functions of Bcl-2 family members and for developing novel therapeutics targeting Bcl-2 mediated apoptotic pathways (Muchmore et al., Nature 381:335-41 (1996)).
The detailed mechanism of Bcl-2 proteins in mediating molecular pathways of apoptosis has been the subject of intensive investigation. It is known that the apoptotic signaling pathway involves the activation of caspases which, once activated, cleave several cellular substrates such as poly(adenosine diphosphate-ribose) polymerase (PARP) and lead to final events of apoptosis. Bcl-2 plays a crucial role in regulating the process of apoptosis. One possible mechanism for Bcl-2 function is that Bcl-2 inhibits the release of cytochrome c from mitochondria. Cytochrome c is important for the activation of caspases. As such, Bcl-2 blocks caspase activation and subsequent events leading to apoptosis.
Being able to block apoptosis, Bcl-2 is known to contribute to neoplastic cell expansion by preventing normal cell turnover caused by physiological cell death mechanisms. High levels and aberrant patterns of Bcl-2 gene expression are found in a wide variety of human cancers, including xcx9c30-60% of prostate, xcx9c90% of colorectal, xcx9c60% of gastric, xcx9c20% of non-small cell lung cancers, xcx9c30% of neuroblastomas, and variable percentages of melanomas, renal cell, and thyroid cancers, as well as acute and chronic lymphocytic and non-lymphocytic leukemias (Ellis et al., Cell Biol. 7, 663 (1991); Henkart, Immunity 1, 343 (1994)); Kxc3xa4gi et al., Science 265, 528 (1994); Kxc3xa4gi et al., Nature 369, 31 (1994); Heusel et al., Cell 76, 977 (1994)).
The expression levels of Bcl-2 protein also correlate with relative resistance to a wide spectrum of current chemotherapeutic drugs and ?-irradiation (Hanada et al., Cancer Res. 53:4978-86 (1993); Kitada et al., Antisense Res. Dev. 4:71-9 (1994); Miyashita et al., Cancer Res. 52:5407-11 (1992); Miyashita et al., Blood 81:151-7 (1993)). Since Bcl-2 can protect against such a wide variety of drugs which have very different mechanisms of action, it is possible that all these drugs use a common final pathway for the eventual induction of cell death which is regulated by Bcl-2. This notion is supported by the findings that chemotherapeutic drugs induce cell death through a mechanism consistent with apoptosis as opposed to necrosis. Therefore, Bcl-2 can inhibit the cell killing effect of currently available anticancer drugs by blocking the apoptotic pathway.
Because of its role in blocking apoptosis, Bcl-2 plays an important role in many types of cancer. As noted above, Bcl-2 blocks apoptosis, thereby preventing normal cell turnover. As a result, neoplastic cell expansion occurs unimpeded by the normal cellular turnover process. Prostate cancer is one particular example where Bcl-2 has important implication in the pathogenesis and treatment for a disease. Approximately 100,000 new cases of prostate cancer are diagnosed each year in the United States and about 30,000 deaths per year are attributable to this disease (Lynn et al., JNCl 87:867 (1995)). It has recently been found that hormone therapy-resistant prostate cancers express Bcl-2 (McDonnell et al., Cancer Res. 52:694-04 (1992)), while the normal prostate cells from which prostate cancers originate lack Bcl-2 (Colombel et al., Am J Pathol 143:390-400 (1993)). This indicates that Bcl-2 may protect prostate cancer cells from undergoing apoptosis induced by the anticancer drugs, such as taxol (Haldar et al., Cancer Res., 56:1235-5 (1996)). The clinical efficacy of nearly every cytotoxic anticancer drug currently available depends directly or indirectly on the assumption that tumor cells grow more rapidly than normal cells. However, this may not apply to human prostate cancer cells, which show very slow growth kinetics. Tumor kinetics studies have indicated that prostate cancer may be the consequence of the imbalance in cell turnover mechanisms more so than an increase in cell cycle rates. Thus, current anticancer drugs may not be effective in eradicating these nonproliferative prostate cancer cells.
The understanding of the biology of Bcl-2 in cancer and chemoresistance has opened new avenues in the development of novel anticancer strategies. One effective approach to overcome the chemoresistance of prostate cancers is to inhibit the protective function of Bcl-2 proteins. New drugs that modulate Bcl-2 mediated apoptotic response would represent a novel mechanism-based strategy for the treatment of prostate cancers and other cancers. Because the function of Bcl-2 is not absolutely necessary in many normal cell types (Veis et al., Cell, 75:229-40 (1993)), a systematic inhibition of Bcl-2 may not affect the normal cellular function. This notion is supported by recent encouraging data from the clinical trial that antisense oligonucleotides targeted against the Bcl-2 gene can specifically inhibit non-Hodgkin""s lymphoma in humans (Webb et al., Lancet 349:1137-41 (1997)). However, the clinical value of such antisense oligonucleotides is limited by their lack of enzymatic stability, cell permeability, and oral activity. As discussed above, currently available anticancer drugs may not be effective due to the chemoresistance of prostate cancer cells. Therefore, there is an impending need for highly potent, cell permeable, and active Bcl-2 inhibitors as a new generation of effective therapeutics for the treatment of prostate cancer, as well as other cancers.
What is needed is methods and agents for enhancing the cell uptake of drugs and biological molecules used as drugs, particular substances used for regulating apoptosis. In particular, what is needed are methods and agents for enhancing the uptake of peptides and proteins used as inhibitors of intracellular targets, so that these molecules may reach their intended intracellular targets, such as the Bcl-2 protein.
It is an object of the invention to provide a novel carrier for transporting chemical and biological agents, particularly proteinaceous molecules, across the cell membrane.
It is an object of the invention to provide conjugates of a carrier substance and a chemical or biological agent, particularly a peptide agent, for delivery into the interior of a cell.
It is an object of the invention to provide novel therapeutics and methods for modulating cell apoptosis, particularly for reversing Bcl-2-mediated blockage of cell apoptosis in cancer cells, virally infected cells and self-reactive lymphocytes.
It is an object of the invention to provide agents to overcome Bcl-2-mediated chemoresistance in tumor cells.
These and other objects of the invention are apparent from the following description.
According to the present invention, a peptide conjugate of formula I
(Rxe2x80x94X)n-peptidexe2x80x83xe2x80x83(I)
is provided wherein:
n is from 1 to 10;
X is
(a) Cxe2x95x90O, when the Rxe2x80x94X group is attached to:
(i) the N-terminus of the peptide, or
(ii) a side chain of the peptide where the functional group of the side chain to which the Rxe2x80x94X group is attached is NH2 or OH; or
(b) O or NH, when the Rxe2x80x94X group is attached to
(i) the C-terminus of the peptide, or
(ii) a side chain of the peptide where the functional group of the side chain to which the Rxe2x80x94X group is attached is COOH or CONH2; and
R is selected from the group consisting of C2-18 alkyl; C2-18 alkoxy; C2-14 alkylenyl containing one or two double bonds;
cyclobutyl; cyclopentyl; cyclohexyl optionally monosubstituted with a C1-5 straight or branched chain alkyl group; phenyl optionally monosubstituted with a C1-5 straight or branched chain alkyl group; and benzyl.
In another embodiment of the invention, a method for modulating apoptosis in cells is provided comprising contacting the cells with a conjugate of a molecule which is a modulator of apoptosis and a moiety of the formula II
(Rxe2x80x94X)nxe2x80x94xe2x80x83xe2x80x83(II)
wherein:
n is from 1 to 10;
X is an atom, chemical bond or chemical group; and
R is as defined above.
When R is alkyl or alkoxy in formulae I or II, the carbon chain may be straight or branched.
Where R is alkyl, it is preferably C3-18 alkyl. According to another preferred embodiment, R is C3-6 branched chain alkyl. Where R is alkylenyl, it is preferably C2-14 alkylenyl containing one double bond or C4-8 alkylenyl containing two double bonds.
Where R is C2-14 alkylenyl containing two double bonds, the bonds may be conjugated or separated.
Preferred substituted phenyl moieties include 2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl and 5-phenylpentyl.
The peptide moiety of the conjugate of the present invention may consist of natural amino acids, or modified or unnatural amino acids. The peptide is typically comprised of L-amino acids, but may contain one or more D-amino acids.
The peptide may be linear or cyclic. The bonds resulting in cyclization may be between the respective N and C termini of amino acids (main chain to main chain connection), from the N or C terminus of one amino acid to the side chain of another amino acid (main chain to side chain), or from the side chain of one amino acid to the side chain of another amino acid (side chain to side chain connection). The peptide may contain a synthetic backbone modification, such as in the case of a peptidomimetic or peptoid.
When n is 1 in formula I, the Rxe2x80x94X group may reside on the peptide C-terminus or N-terminus, or may reside on the side-chain of an amino acid residue. When n is greater than 1 in formula I, the Rxe2x80x94X groups can be attached on any positions of the peptide. Preferably, the Rxe2x80x94X group is attached to the N-terminus of the peptide, or to the side chain of an amino acid residue.
The invention is also directed to a method for enhancing the cellular uptake of a peptide comprising conjugating said peptide to a carrier moiety (Rxe2x80x94X)nxe2x80x94, to form a conjugate as described above.
In one embodiment, the conjugate comprises a peptide which is an inhibitor of the function of an intracellular biological target, such as Bcl-2. According to one such embodiment, the peptide binds to the Bcl-2 protein.
A method is provided for reversing Bcl-2-mediated blockage of apoptosis in cancer cells comprising contacting said cells with a conjugate comprising a peptide which is an inhibitor of the function of Bcl-2.
A method is provided for treating a subject afflicted with a cancer characterized by cancer cells which express Bcl-2. The method comprises administering to the subject an effective amount of a conjugate which comprises a peptide which is an inhibitor of the function of Bcl-2.
In another embodiment of the invention, a conjugate comprises an exogenous molecule, not limited to a peptide, which is a modulator of apoptosis. The modulator is conjugated to a carrier group, (Rxe2x80x94Xxe2x80x94)nxe2x80x94, as defined above. The modulator may comprise any substance which has the effect of either inducing or inhibiting apoptosis in the target cells. The modulator may comprise a peptide, polypeptide, protein, oligonucleotide, polynucleotide, glycoprotein, oligosaccharide, amino acid, nucleoside, nucleotide, or any other organic molecule which has a modulating effect on apoptosis in cells, particularly cells which are not otherwise permeable to the modulator, absent conjugation to the carrier (Rxe2x80x94X)n as described above.
By xe2x80x9cmodulator of apoptosisxe2x80x9d is meant a substance which either inhibits or induces apoptosis in a cell. By xe2x80x9capoptosisxe2x80x9d or xe2x80x9capoptotic deathxe2x80x9d is meant the programed death which results in controlled autodigestion of the cell, as opposed to necrotic cell death. Apoptotic cell death is characterized by cytoskelet al disruption, cell shrinkage, and membrane blebbing. The nucleus undergoes condensation and nuclear DNA becomes degraded and fragmented. Apoptosis is also characterized by loss of mitochondrial function. Necrotic cell death, on the other hand, is a pathological form of cell death resulting from acute cellular injury, which is typified by rapid swelling and lysis.
According to certain embodiments of the invention, the modulator is an inhibitor of apoptosis, and the target cells induced to undergo apoptosis comprise cancer cells, virus-infected cells or self-reactive lymphocytes. Thus, the conjugates of the invention may be used to treat cancer, viral infection, or autoimmune disorders.
The nomenclature used to describe polypeptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by a three-letter or one letter designation, corresponding to the trivial name of the amino acid, in accordance with the following schedule:
The following definitions, of terms used throughout the specification, are intended as an aid to understanding the scope and practice of the present invention.
A xe2x80x9cpeptidexe2x80x9d is a compound comprised of amino acid residues covalently linked by peptide bonds. Peptides comprising a large number of amino acids are sometimes called xe2x80x9cpolypeptidesxe2x80x9d. The expression xe2x80x9cpeptidesxe2x80x9d is understood to include xe2x80x9cpolypeptidesxe2x80x9d as well as proteins. Further included in the scope of xe2x80x9cpeptidexe2x80x9d as used herein are synthetic variants thereof including various backbone modifications, such as the molecules known as peptidomimetics and peptoids. Further included in the scope of xe2x80x9cpeptidexe2x80x9d as used herein are variants which include alterations of amino acid side chains, including but not limited to attachment of carbohydrate moieties.
The expression xe2x80x9camino acidxe2x80x9d as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. xe2x80x9cNatural amino acidxe2x80x9d means any of the twenty primary, naturally occurring amino acids which typically form peptides, polypeptides, and proteins. xe2x80x9cSynthetic amino acidxe2x80x9d means any other amino acid, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, xe2x80x9csynthetic amino acidxe2x80x9d also encompasses chemically modified amino acids, including but not limited to salts, derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change the peptide""s circulating half life without adversely affecting their activity. Additionally, a disulfide linkage may be present or absent in the peptides of the invention.
Amino acids have the following general structure: 
Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. The amino acids of the peptides described herein and in the appended claims are understood to be either D or L amino acids with L amino acids being preferred.